This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During the assembly of many bacteriophages and some eukaryotic viruses, such as herpesviruses and some adenoviruses, the nucleic acid is inserted into a preformed capsid precursor through a unique portal vertex. In the double-stranded DNA viruses the protein complex actively involved in nucleic acid packaging is called terminase and usually consists of two subunits. The smaller subunit is responsible for both the recognition of a unique site in the viral nucleic acid (pac site) and the initiation of terminase complex formation. The large subunit possesses the necessary enzymatic activities for packaging, namely ATPase activity to provide energy for DNA translocation and endonuclease activity for cleavage of DNA at the beginning and end of the packaging event. Molecular mechanisms of terminase complex assembly and DNA packaging are not understood in detail for any virus. Identification of structure/function relationships for terminase subunits has been particularly elusive. Interaction of the small subunit with its DNA recognition site is crucial for the initiation of productive packaging and eventual virus maturation. The small subunit also binds to the large subunit and stimulates its ATPase activity. Knowledge of the three-dimensional structure of the small subunit would significantly advance our understanding of terminase complex assembly and initiation of packaging. Such structural information will also enhance the interpretation of data already collected in our laboratory using complementary techniques (Raman spectroscopy, CD spectroscopy, sedimentation equilibria, etc.). The structure of gp3 would also provide valuable assistance in proposed future investigations of gp3/DNA binding involving the pac site. In our ongoing studies of the small and large (gp2) terminase subunits of bacteriophage P22, we have developed procedures for efficient purification of both to better than 99% homogeneity. We have found recently that the small subunit oligomerizes into a symmetrical decameric ring that is able to bind to dsDNA. We have visualized the decamer from averaging negatively stained transmission electron micrographs. The image shown in Figure 1 was refined from ~450 aligned particles by use of a reference independent method and by using translation and rotation operations without any symmetry assumption for the alignment. The ring exhibits ten-fold symmetry with a central hole of ~2 nm diameter and ten outer spikes. The outer diameter of the ring is about 11.2 nm. Although ring formation has been proposed for small terminase subunits of bacteriophages SPP1 and T4, the oligomeric states have been speculative and no structural details have been revealed. Bioinformatics and molecular biology experiments on these phages also suggest important roles for the small subunit in terminase assembly and function.